Ornella Maglio

An artificial di-iron oxo-protein with phenol oxidase activity.

Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site.

Discovering protein secondary structures: Classification and description of isolated α-turns

Irregular protein secondary structures are believed to be important structural domains involved in molecular recognition processes between proteins, in interactions between peptide substrates and receptors, and in protein folding. In these respects tight turns are being studied in detail. They also represent template structures for the design of new molecules such as drugs, pesticides, or antigens. Isolated α-turns, not participating in α-helical structures, have received little attention due to the overwhelming presence of other types of tight turns in peptide and protein structures. The growing number of protein X-ray structures allowed us to undertake a systematic search into the Protein Data Bank of this uncharacterized protein secondary structure. A classification of isolated α-turns into different types, based on conformational similarity, is reported here. A preliminary analysis on the occurrence of some particular amino acids in certain positions of the turned structure is also presented.

Artificial diiron proteins: From structure to function†

De novo protein design provides an attractive approach for the construction of models to probe the features required for the function of complex metalloproteins. These minimal models contain the essential elements believed necessary for activity of the protein. In this article, we summarize the design, structure determination, and functional properties of a family of artificial diiron proteins.

Preorganization of molecular binding sites in designed diiron proteins

De novo protein design provides an attractive approach to critically test the features that are required for metalloprotein structure and function. Previously we designed and crystallographically characterized an idealized dimeric model for the four-helix bundle class of diiron and dimanganese proteins [Dueferri 1 (DF1)]. Although the protein bound metal ions in the expected manner, access to its active site was blocked by large bulky hydrophobic residues. Subsequently, a substrate-access channel was introduced proximal to the metal-binding center, resulting in a protein with properties more closely resembling those of natural enzymes. Here we delineate the energetic and structural consequences associated with the introduction of these binding sites. To determine the extent to which the binding site was preorganized in the absence of metal ions, the apo structure of DF1 in solution was solved by NMR and compared with the crystal structure of the di-Zn(II) derivative. The overall fold of the apo protein was highly similar to that of the di-Zn(II) derivative, although there was a rotation of one of the helices. We also examined the thermodynamic consequences associated with building a small molecule-binding site within the protein. The protein exists in an equilibrium between folded dimers and unfolded monomers. DF1 is a highly stable protein (Kdiss = 0.001 fM), but the dissociation constant increases to 0.6 nM (ΔΔG = 5.4 kcal/mol monomer) as the active-site cavity is increased to accommodate small molecules.

Structure-based design of an urokinase-type plasminogen activator receptor–derived peptide inhibiting cell migration and lung metastasis

The urokinase-type plasminogen activator receptor (uPAR) plays a central role in sustaining the malignant phenotype and promoting tumor metastasis. The Ser88-Arg-Ser-Arg-Tyr92 is the minimum chemotactic sequence of uPAR required to induce the same intracellular signaling as its ligand uPA. Here, we describe the generation of new peptide inhibitors of cell migration and invasion derived from SRSRY by a drug design approach. Ac-Arg-Glu-Arg-Phe-NH2 (i.e., RERF), which adopts a turned structure in solution, was selected for its ability to potently prevent SRSRY-directed cell migration. Fluorescein-RERF associates with very high affinity to RBL-2H3 rat basophilic leukemia cells expressing the human formyl peptide receptor (FPR). Accordingly, femtomolar concentrations of RERF prevent agonist-dependent internalization of FPR and inhibit N-formyl-Met-Leu-Phe–dependent migration in a dose-dependent manner. In the absence of FPR, fluorescein-RERF binds to cell surface at picomolar concentrations in an αv integrin–dependent manner. The involvement of vitronectin receptor is further supported by the findings that 100 pmol/L RERF selectively inhibits vitronectin-dependent RBL-2H3 cell migration and prevents SRSRY-triggered uPAR/αv association. Furthermore, RERF reduces the speed of wound closure and the extent of Matrigel invasion by human fibrosarcoma HT1080 cells without affecting cell proliferation. Finally, a 3- to 5-fold reduction of lung metastasis number and size in nude mice following i.v. injection of green fluorescent protein–expressing HT1080 cells in the presence of 3.32 mg/kg RERF is observed. Our findings indicate that RERF effectively prevents malignant cell invasion in vivo with no signs of toxicity and may represent a promising prototype drug for anticancer therapy. [Mol Cancer Ther 2009;8(9):2708–17]

Artificial di-iron proteins: solution characterization of four helix bundles containing two distinct types of inter-helical loops

Peptide-based models have an enormous impact for the development of metalloprotein models, as they seem appropriate candidates to mimic both the structural characteristics and reactivity of the natural systems. Through the de novo design of four-helix bundles, we developed the DF (Due Ferri) family of artificial proteins, as models of di-iron and di-manganese metalloproteins. The goal of our research is to elucidate how the electrostatic environment, polarity and solvent accessibility of the metal-binding site, influence the functional properties of di-iron proteins. The first two subsets of the DF protein family, DF1 and DF2, consist of two non-covalently associated helix-loop-helix motifs, which bind the di-metal cofactor near the center of the structure. The DF2 subset was designed to improve the properties of DF1: DF2 and DF2t have several changes in their sequences to improve solubility and metal ion access, as well as a change in the loop connecting the two helices. In order to evaluate how these changes affect the overall structure of the model proteins, we solved the NMR structures of the di-Zn(II) complexes of DF2 and DF2t, and compared these structures with those recently obtained from X-ray crystallography. Further, we examined the thermodynamic consequences associated with the mutations, by measuring the stability of DF2t in the presence of different metal ions, and comparing the results with the data already obtained for DF2. Taken together, analysis of all the data showed the importance of the turn conformation in the design and stability of four-helix bundle.

An urokinase receptor antagonist that inhibits cell migration by blocking the formyl peptide receptor

Urokinase receptor (uPAR) plays a key role in physiological and pathological processes sustained by an altered cell migration. We have developed peptides carrying amino acid substitutions along the Ser88‐Arg‐Ser‐Arg‐Tyr92 (SRSRY) uPAR chemotactic sequence. The peptide pyro glutamic acid (pGlu)‐Arg‐Glu‐Arg‐Tyr‐NH2 (pERERY‐NH2) shares the same binding site with SRSRY and competes with N‐formyl‐Met‐Leu‐Phe (fMLF) for binding to the G‐protein‐coupled N‐formyl‐peptide receptor (FPR). pERERY‐NH2 is a dose‐dependent inhibitor of both SRSRY‐ and fMLF‐directed cell migration, and prevents agonist‐induced FPR internalization and fMLF‐dependent ERK1/2 phosphorylation. pERERY‐NH2 is a new and potent uPAR inhibitor which may suggest the generation of new pharmacological treatments for pathological conditions involving increased cell migration.

Miniaturized heme proteins: crystal structure of Co(III)-mimochrome IV

Protein design provides an attractive approach to test the essential features required for folding and function. Previously, we described the design and structural characterization in solution of mimochromes, a series of miniaturized metalloproteins, patterned after the F-helix of the hemoglobin β-chain. Mimochromes consist of two medium-sized helical peptides, covalently linked to the deuteroporphyrin. CD and NMR characterization of the prototype, mimochrome I, revealed that the overall structure conforms well to the design. However, formation of Δ and Λ diastereomers was observed. To overcome the problem of diastereomer formation, we re-designed mimochrome I, by engineering intramolecular, interchain interactions. The resulting model was mimochrome IV: the solution structural characterization showed the presence of the Λ isomer as a unique form. To examine the extent to which the stereochemical stability and uniqueness of mimochrome IV was retained in the solid state, the crystal structure of Co(III)-mimochrome IV was solved by X-ray diffraction, and compared to the solution structure of the same derivative. Co(III)-mimochrome IV structures, both in solution and in the solid state, are characterized by the following common features: a bis-His axial coordination, a Λ configuration around the metal ion, and a predominant helical conformation of the peptide chains. However, in the crystal structure, intrachain Glu1–Arg9 ion pairs are preferred over the designed, and experimentally found in solution, interchain interactions. This ion pairing switch may be related to strong packing interactions.

De Novo Design, Synthesis and Characterisation of MP3, A New Catalytic Four‐Helix Bundle Hemeprotein

A new artificial metalloenzyme, MP3 (MiniPeroxidase 3), designed by combining the excellent structural properties of four-helix bundle protein scaffolds with the activity of natural peroxidases, was synthesised and characterised. This new hemeprotein model was developed by covalently linking the deuteroporphyrin to two peptide chains of different compositions to obtain an asymmetric helix-loop-helix/heme/helix-loop-helix sandwich arrangement, characterised by 1) a His residue on one chain that acts as an axial ligand to the iron ion; 2) a vacant distal site that is able to accommodate exogenous ligands or substrates; and 3) an Arg residue in the distal site that should assist in hydrogen peroxide activation to give an HRP-like catalytic process. MP3 was synthesised and characterised as its iron complex. CD measurements revealed the high helix-forming propensity of the peptide, confirming the appropriateness of the model procedure; UV/Vis, MCD and EPR experiments gave insights into the coordination geometry and the spin state of the metal. Kinetic experiments showed that Fe(III)-MP3 possesses peroxidase-like activity comparable to R38A-hHRP, highlighting the possibility of mimicking the functional features of natural enzymes. The synergistic application of de novo design methods, synthetic procedures, and spectroscopic characterisation, described herein, demonstrates a method by which to implement and optimise catalytic activity for an enzyme mimetic.

Molecular engineering of RANTES peptide mimetics with potent anti-HIV-1 activity

The chemokine receptor CCR5 is utilized as a critical coreceptor by most primary HIV‐1 strains. While the lack of structural information on CCR5 has hampered the rational design of specific inhibitors, mimetics of the chemokines that naturally bind CCR5 can be molecularly engineered. We used a structure‐guided approach to design peptide mimetics of the N‐loop and βl‐strand regions of regulated on activation normal T‐cell‐expressed and secreted (RANTES)/CCL5, which contain the primary molecular determinants of HIV‐1 blockade. Rational modifications were sequentially introduced into the N‐loop/βl‐strand sequence, leading to the generation of mimetics with potent activity against a broad spectrum of CCR5‐specific HIV‐1 isolates (IC50 range: 104–640 nM) but lacking activity against CXCR4‐specific HIV‐1 isolates. Functional enhancement was initially achieved with the stabilization of the N loop in the β‐extended conformation adopted in full‐length RANTES, as confirmed by nuclear magnetic resonance (NMR) analysis. However, the most dramatic increase in antiviral potency resulted from the engraftment of an in siZico‐optimized linker segment designed using de novo structure‐prediction algorithms to stabilize the C‐terminal α‐helix and experimentally validated by NMR. Our mimetics exerted CCR5‐antagonistic effects, demonstrating that the antiviral and proinflammatory functions of RANTES can be uncoupled. RANTES peptide mimetics provide new leads for the development of safe and effective HIV‐1 entry inhibitors.—Lusso, P., Vangelista, L., Cimbro, R., Secchi, M., Sironi, F., Longhi, R., Faiella, M., Maglio, O., Pavone, V. Molecular engineering of RANTES peptide mimetics with potent anti‐HIV‐1 activity. FASEB J. 25, 1230–1243 (2011). www.fasebj.org